Search results for: endothelium

Authors: Yousuf Al Suleimani, Ahmed Al Mahruqi

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A typical cannabinoid O-1602 induces vasorelaxation and activates the orphan G protein-coupled receptor GPR55 in human endothelial cells. The aim of this study is to characterize the mechanisms of endothelium-independent relaxation of O-1602 in the rat small mesenteric artery using wire myograph. In endothelium-denuded vessels, O-1602 partially produced concentration-dependent vasorelaxation. In vessels depleted of intracellular Ca2+ (by EGTA and methoxamine), CaCl2 produced concentration-dependent contraction. Preincubation with O-1602 (at 10 µM and 30 µM) abolished the contractile responses (P<0.01). The putative antagonist at novel “endothelial anandamide receptor” O-1918 (10 µM) significantly reversed the inhibitory effect of O-1602 on CaCl2-induced vasoconstriction. It is likely that the mechanism of endothelium-independent vasorelaxation to O-1602 is mediated by interfering with Ca2+ entry via an O-1918-sensitive pathway.

Keywords: O-1602, endothelium, vasorelaxation, calcium

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Authors: Majid Moshirfar, Kyle Margulies, Yasmyne C. Ronquillo, Phillip Hoopes

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A total of 66 articles were reviewed to compare glaucoma and its associated surgeries' effect on central corneal endothelium cell density (CECD). The paper reports the average reported central CECD loss at 3-, 6-, 12-, 24-, 36-, 48-, and 60-month post-operation for each glaucoma surgery. ALT, MLT, SLT, CS AGV, VC BGI, Hydrus + phaco, XEN gel + phaco, PRESERFLO, Dual iStent, or Trabectome had no significant impact on postoperative CECD compared to either preoperative CECD or control group CECD. The highest CECD loss was found to be EXPRESS-phaco, AC AGV, CS BGI, CS BGI, AC BGI, and AC BGI at the 3-, 6-, 12-, 24-, 36-, 48-, and 60-month follow-ups, respectively. AC AGV, Trab + MMC, Trab, AC BGI, Trab + MMC, Cypass, and Cypass showed the smallest reduction of CECD at the 3-, 6-, 12-, 24-, 36-, 48-, and 60-month follow-ups.

Keywords: glaucoma, corneal endothelium, cell density, surgery outcome

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Authors: Ammar Boudaka, Maryam Al-Suleimani, Hajar BaOmar, Intisar Al-Lawati, Fahad Zadjali

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Background: Hypertension is increasingly becoming a matter of medical and public health importance. The maintenance of normal blood pressure requires a balance between cardiac output and total peripheral resistance. The endothelium, through the release of vasodilating factors, plays an important role in the control of total peripheral resistance and hence blood pressure homeostasis. Transient Receptor Potential Vanilloid type 4 (TRPV4) is a mechanosensitive non-selective cation channel that is expressed on the endothelium and contributes to endothelium-mediated vasodilation. So far, no data are available about the morphological and functional status of this channel in hypertensive cases. Objectives: This study aimed to investigate whether there is any difference in the morphological and functional features of TRPV4 in the mesenteric artery of normotensive and hypertensive rats. Methods: Functional feature of TRPV4 in four experimental animal groups: young and adult Wistar-Kyoto rats (WKY-Y and WKY-A), young and adult spontaneously hypertensive rats (SHR-Y and SHR-A), was studied by adding 5 µM 4αPDD (TRPV4 agonist) to mesenteric arteries mounted in a four-chamber wire myograph and pre-contracted with 4 µM phenylephrine. The 4αPDD-induced response was investigated in the presence and absence of 1 µM HC067047 (TRPV4 antagonist), 100 µM L-NAME (nitric oxide synthase inhibitor), and endothelium. The morphological distribution of TRPV4 in the wall of rat mesenteric arteries was investigated by immunostaining. Real-time PCR was used in order to investigate mRNA expression level of TRPV4 in the mesenteric arteries of the four groups. The collected data were expressed as mean ± S.E.M. with n equal to the number of animals used (one vessel was taken from each rat). To determine the level of significance, statistical comparisons were performed using the student’s t-test and considered to be significantly different at p<0.05. Results: 4αPDD induced a relaxation response in the mesenteric arterial preparations (WKY-Y: 85.98% ± 4.18; n = 5) that was markedly inhibited by HC067047 (18.30% ± 2.86; n= 5; p<0.05), endothelium removal (19.93% ± 1.50; n = 5; p<0.05) and L-NAME (28.18% ± 3.09; n = 5; p<0.05). The 4αPDD-induced relaxation was significantly lower in SHR-Y compared to WKY-Y (SHR-Y: 70.96% ± 3.65; n = 6, WKY-Y: 85.98% ± 4.18; n = 5-6, p<0.05. Moreover, the 4αPDD-induced response was significantly lower in WKY-A than WKY-Y (WKY-A: 75.58 ± 1.30; n = 5, WKY-Y: 85.98% ± 4.18; n = 5, p<0.05). Immunostaining study showed immunofluorescent signal confined to the endothelial layer of the mesenteric arteries. The expression of TRPV4 mRNA in SHR-Y was significantly lower than in WKY-Y (SHR-Y; 0.67RU ± 0.34; n = 4, WKY-Y: 2.34RU ± 0.15; n = 4, p<0.05). Furthermore, TRPV4 mRNA expression in WKY-A was lower than its expression in WKY-Y (WKY-A: 0.62RU ± 0.37; n = 4, WKY-Y: 2.34RU ± 0.15; n = 4, p<0.05). Conclusion: Stimulation of TRPV4, which is expressed on the endothelium of rat mesenteric artery, triggers an endothelium-mediated relaxation response that markedly decreases with hypertension and growing up changes due to downregulation of TRPV4 expression.

Keywords: hypertension, endothelium, mesenteric artery, TRPV4

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Authors: Sahar M. El-Gowilly, Mai M. Helmy, Hanan M. El-Gowelli

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Methotrexate (MTX) is widely used in treatment of cancers and autoimmune diseases. However, nephrotoxicity is one of the most important side effects of MTX. The peroxisome proliferator-activated receptor gamma agonist, pioglitazone (PIO), is known to exert anti-inflammatory and reno-protective effects in various kidney injuries. The purpose of this study was to investigate the potential involvement of endothelial damage in MTX-induced renal injury and to elaborate the possible protective effect of PIO against MTX-induced nephropathy. Compared with saline-treated rats, treatment with MTX (7 mg/kg for 3 day) caused significant elevations in serum levels of urea and creatinine, increased renal nitrate/nitrite level and impaired renovascular responsiveness of isolated perfused kidney to endothelium-dependent vasodilations induced by acetylcholine (0.01-2.43 nmol) and isoprenaline (1µmol). These effects were abolished by concurrent treatment with PIO (2.5 mg/kg, for 5 days starting two days before MTX). Alternatively, MTX treatment did not affect endothelium-independent renovascular relaxation induced by sodium nitroprusside (1-30 μmole). The possibility that alterations in renal antioxidants, circulating cytokine and apoptotic factor (Fas) levels contributed to MTX-PIO interaction was assessed. PIO treatment abrogated renal oxidative stress (decreased reduced glutathione and catalase activity and increased malondialdehyde), elevated serum cytokine (interleukin-6, interleukin-10, tumor necrosis factor-alpha and transforming growth factor-beta1) and Fas induced by MTX. Histologically, MTX caused defused tubular cells swelling and vacuolization associated with endothelial damage in renal arterioles. These effects disappeared upon co-treated with PIO. Collectively, PIO abolished MTX-induced endothelium dysfunction and nephrotoxicity via ameliorating oxidative stress and rectifying cytokines and Fas abnormalities caused by MTX.

Keywords: methotrexate, pioglitazone, endothelium, kidney

Procedia PDF Downloads 280

Authors: Sahar M. El-Gowilly, Mai M. Helmy, Hanan M. El-Gowelli

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Methotrexate (MTX) is widely used in treatment of cancers and autoimmune diseases. However, nephrotoxicity is one of its most important side effects. The peroxisome proliferator-activated receptor gamma agonist, pioglitazone, is known to exert antiinflammatory and reno-protective effects in various kidney injuries. The purpose of this study was to investigate the potential involvement of endothelial damage in MTX-induced renal injury and to elaborate the possible protective effect of pioglitazone against MTX-induced endothelial impairment. Compared with saline-treated rats, treatment with MTX (7 mg/kg for 3 day) caused significant elevations in serum levels of urea and creatinine, increased renal nitrate/nitrite level and impaired renovascular responsiveness of isolated perfused kidney to endothelium-dependent vasodilations induced by acetylcholine (0.01-2.43 nmol) and isoprenaline (1µmol). These effects were abolished by concurrent treatment with pioglitazone (2.5 mg/kg, for 5 days starting two days before MTX). Alternatively, MTX treatment did not affect endothelium-independent renovascular relaxation induced by sodium nitroprusside (0.001-10 μmole). The possibility that alterations in renal antioxidants, circulating cytokine and apoptotic factor (Fas) levels contributed to MTX-pioglitazone interaction was assessed. Pioglitazone treatment abrogated renal oxidative stress (decreased reduced glutathione and catalase activity and increased malondialdehyde), elevated serum cytokine (interleukin-6, interleukin-10, tumor necrosis factor-alpha and transforming growth factor-beta1) and Fas induced by MTX. Histologically, MTX caused defused tubular cells swelling and vacuolization associated with endothelial damage in renal arterioles. These effects disappeared upon co-treated with pioglitazone. Collectively, pioglitazone abolished MTX-induced endothelium dysfunction and nephrotoxicity via ameliorating oxidative stress and rectifying cytokines and Fas abnormalities caused by MTX.

Keywords: methotrexate, pioglitazone, endothelium, kidney

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Authors: M. P. S. Tomar, Neelam Bansal

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Histomorphological, histochemical and scanning electron microscopic observations were recorded in developing cornea of buffalo fetuses. The samples from fetal cornea were collected in appropriate fixative from slaughter house and Veterinary Clinics, GADVASU, Ludhiana. The microscopic slides were stained for detailed histomorphological and histochemical studies. The scanning electron microscopic studies were performed at Electron microscopy & Nanobiology Lab, PAU Ludhiana. In present study, it was observed that, in 36 days (d) fetus, the corneal epithelium was well marked single layered structure which was placed on stroma mesenchyme. Cornea appeared as the continuation of developing sclera. The thickness of cornea and its epithelium increased as well as the epithelium started becoming double layered in 47d fetus at corneo-scleral junction. The corneal thickness in this stage suddenly increased thus easily distinguished from developing sclera. The separation of corneal endothelium from stroma was evident as a single layered epithelium. The stroma possessed numerous fibroblasts in 49d stage eye. Descemet’s membrane was appeared at 52d stage. The limbus area was separated by a depression from the developing cornea in 61d stage. In 65d stage, the Bowman’s layer was more developed. Fibroblasts were arranged parallel to each other as well as parallel to the surface of developing cornea in superficial layers. These fibroblasts and fibers were arranged in wavy pattern in the center of stroma. Corneal epithelium started to be stratified as a double layered epithelium was present in this age of fetal eye. In group II (>120 Days), the corneal epithelium was stratified towards a well marked irido-corneal angle. The stromal fibroblasts followed a complete parallel arrangement in its entire thickness. In full term fetuses, a well developed cornea was observed. It was a fibrous layer which had five distinct layers. From outside to inwards were described as the outer most layer was the 7-8 layered corneal epithelial, subepithelial basement membrane (Bowman’s membrane), substantia propria or stroma, posterior limiting membrane (Descemet’s membrane) and the posterior epithelium (corneal endothelium). The corneal thickness and connective tissue elements were continued to be increased. It was 121.39 + 3.73µ at 36d stage which increased to 518.47 + 4.98 µ in group III fetuses. In fetal life, the basement membrane of corneal epithelium and endothelium depicted strong to intense periodic Acid Schiff’s (PAS) reaction. At the irido-corneal angle, the endothelium of blood vessels was also positive for PAS activity. However, cornea was found mild positive for alcian blue reaction. The developing cornea showed strong reaction for basic proteins in outer epithelium and the inner endothelium layers. Under low magnification scanning electron microscope, cornea showed two types of cells viz. light cells and dark cells. The light cells were smaller in size and had less number of microvilli in their surface than in the dark cells. Despite these surface differences between light and dark cells, the corneal surface showed the same general pattern of microvilli studding all exposed surfaces out to the cell margin. which were long (with variable height), slight tortuous slender and possessed a micro villus shaft with a very prominent knob.

Keywords: buffalo, cornea, eye, fetus, ontogeny, scanning electron microscopy

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Authors: Michelle Maurer, Antonia Last, Mark S. Gresnigt, Bernhard Hube, Alexander S. Mosig

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The intestinal epithelium forms an essential barrier to prevent translocation of microorganisms, toxins or other potentially harmful molecules into the bloodstream. In particular, dendritic cells of the intestinal epithelium orchestrate an adapted response of immune tolerance to commensals and immune defense against invading pathogens. Systemic inflammation is typically associated with a dysregulation of this adapted immune response and is accompanied by a disruption of the epithelial and endothelial gut barrier which enables dissemination of pathogens within the human body. To understand the pathophysiological mechanisms underlying the inflammation-associated gut barrier breakdown, it is crucial to elucidate the complex interplay of the host and the intestinal microbiome. A microfluidically perfused three-dimensional intestine-on-chip model was established to emulate these processes in the presence of immune cells, commensal bacteria, and facultative pathogens. Multi-organ tissue flow (MOTiF) biochips made from polystyrene were used for microfluidic perfusion of the intestinal tissue model. The biochips are composed of two chambers separated by a microporous membrane. Each chamber is connected to inlet and outlet channels allowing independent perfusion of the individual channels and application of microfluidic shear stress. Human umbilical vein endothelial cells (HUVECs), monocyte-derived macrophages and intestinal epithelial cells (Caco-2) were assembled on the biochip membrane. Following 7 – 14 days of growth in the presence of physiological flow conditions, the epithelium was colonized with the commensal bacterium Lactobacillus rhamnosus, while the endothelium was perfused with peripheral blood mononuclear cells (PBMCs). Additionally, L. rhamnosus was co-cultivated with the opportunistic fungal pathogen Candida albicans. Within one week of perfusion, the epithelial cells formed self-organized and well-polarized villus- and crypt-like structures that resemble essential morphological characteristics of the human intestine. Dendritic cells were differentiated in the epithelial tissue that specifically responds to bacterial lipopolysaccharide (LPS) challenge. LPS is well-tolerated at the luminal epithelial side of the intestinal model without signs of tissue damage or induction of an inflammatory response, even in the presence of circulating PBMC at the endothelial lining. In contrast, LPS stimulation at the endothelial side of the intestinal model triggered the release of pro-inflammatory cytokines such as TNF, IL-1β, IL-6, and IL-8 via activation of macrophages residing in the endothelium. Perfusion of the endothelium with PBMCs led to an enhanced cytokine release. L. rhamnosus colonization of the model was tolerated in the immune competent tissue model and was demonstrated to reduce damage induced by C. albicans infection. A microfluidic intestine-on-chip model was developed to mimic a systemic infection with a dysregulated immune response under physiological conditions. The model facilitates the colonization of commensal bacteria and co-cultivation with facultative pathogenic microorganisms. Both, commensal bacteria alone and facultative pathogens controlled by commensals, are tolerated by the host and contribute to cell signaling. The human intestine-on-chip model represents a promising tool to mimic microphysiological conditions of the human intestine and paves the way for more detailed in vitro studies of host-microbiota interactions under physiologically relevant conditions.

Keywords: host-microbiota interaction, immune tolerance, microfluidics, organ-on-chip

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Authors: Ryan D. Martinus

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Inflammation of the endothelium is an important process leading to diabetic atherosclerosis. However, the molecular mechanisms by which diabetes contributes to endothelial inflammation remain to be established. Using In-vitro cultured Human cells and Hsp60 specific ELISA assays, we show that Hsp60 is not only induced in Human monocyte cells under hyperglycaemic conditions but that the Hsp60 is also secreted from these cells. Furthermore, we also demonstrate that the Hsp60 secreted from these monocyte cells is also able to activate Toll-like receptor-4 (TLR4) from Human endothelial cells. This suggests that a potential link may exist between the hyperglycaemia-induced expression of Hsp60 in monocyte cells and vascular inflammation. Circulating levels of Hsp60 due to mitochondrial stress in diabetes patients could, therefore, be an important modulator of inflammation in endothelial cells and thus contribute to the increased incidences of atherosclerosis in diabetes mellitus.

Keywords: mitochondria, Hsp60, inflammation, diabetes mellitus

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Authors: Chirag Raval, Jimmy Toussaint, Tieuvi Nguyen, Hadi Fadaifard, George Wolberg, Steven Quarfordt, Kung-ming Jan, David S. Rumschitzki

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Numerous studies examine aquaporins’ role in osmotic water transport in various systems but virtually none focus on aquaporins’ role in hydrostatically-driven water transport involving mammalian cells save for our laboratory’s recent study of aortic endothelial cells. Here we investigate aquaporin-1 expression and function in the aortic endothelium in two high-renin rat models of hypertension, the spontaneously hypertensive genomically altered Wystar-Kyoto rat variant and Sprague-Dawley rats made hypertensive by two kidney, one clip Goldblatt surgery. We measured aquaporin-1 expression in aortic endothelial cells from whole rat aortas by quantitative immunohistochemistry, and function by measuring the pressure driven hydraulic conductivities of excised rat aortas with both intact and denuded endothelia on the same vessel. We use them to calculate the effective intimal hydraulic conductivity, which is a combination of endothelial and subendothelial components. We observed well-correlated enhancements in aquaporin-1 expression and function in both hypertensive rat models as well as in aortas from normotensive rats whose expression was upregulated by 2h forskolin treatment. Upregulated aquaporin-1 expression and function may be a response to hypertension that critically determines conduit artery vessel wall viability and long-term susceptibility to atherosclerosis. Numerous studies examine aquaporins’ role in osmotic water transport in various systems but virtually none focus on aquaporins’ role in hydrostatically-driven water transport involving mammalian cells save for our laboratory’s recent study of aortic endothelial cells. Here we investigate aquaporin-1 expression and function in the aortic endothelium in two high-renin rat models of hypertension, the spontaneously hypertensive genomically altered Wystar-Kyoto rat variant and Sprague-Dawley rats made hypertensive by two kidney, one clip Goldblatt surgery. We measured aquaporin-1 expression in aortic endothelial cells from whole rat aortas by quantitative immunohistochemistry, and function by measuring the pressure driven hydraulic conductivities of excised rat aortas with both intact and denuded endothelia on the same vessel. We use them to calculate the effective intimal hydraulic conductivity, which is a combination of endothelial and subendothelial components. We observed well-correlated enhancements in aquaporin-1 expression and function in both hypertensive rat models as well as in aortas from normotensive rats whose expression was upregulated by 2h forskolin treatment. Upregulated aquaporin-1 expression and function may be a response to hypertension that critically determines conduit artery vessel wall viability and long-term susceptibility to atherosclerosis.

Keywords: acute hypertension, aquaporin-1, hydraulic conductivity, hydrostatic pressure, aortic endothelial cells, transcellular flow

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Authors: Forough Ataollahi, Sumit Pramanik, Belinda Pingguan-Murphy, Wan Abu Bakar Bin Wan Abas, Noor Azuan Bin Abu Osman

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Endothelium proliferation is an important process in cardiovascular homeostasis and can be regulated by extracellular environment, as cells can actively sense mechanical environment. In this study, we evaluated endothelial cell proliferation on PDMS/alumina (Al2O3) composites and pure PDMS. The substrates were prepared from pure PDMS and its composites with 5% and 10% Al2O3 at curing temperature 50˚C for 4 h and then characterized by mechanical, structural and morphological analyses. Higher stiffness was found in the composites compared to the pure PDMS substrate. Cell proliferation of the cultured bovine aortic endothelial cells on substrate materials were evaluated via Resazurin assay and 1, 1’-Dioctadecyl-1, 3, 3, 3’, 3’-Tetramethylindocarbocyanine Perchlorate-Acetylated LDL (Dil-Ac-LDL) cell staining, respectively. The results revealed that stiffer substrates promote more endothelial cells proliferation to the less stiff substrates. Therefore, this study firmly hypothesizes that the stiffness elevates endothelial cells proliferation.

Keywords: stiffness, proliferation, bovine aortic endothelial cells, extra cellular matrix, vascular

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Authors: Rania Abdulkareem Aboubakr Mahdaly Ammar

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The aortic valve (AV) is a complex mechanical environment that includes flexure, tension, pressure and shear stress forces to blood flow during cardiac cycle. This mechanical environment regulates AV tissue structure by constantly renewing and remodeling the phenotype. In vitro, ex vivo and in vivo studies have explained that pathological states such as hypertension and congenital defects like bicuspid AV ( BAV ) can potentially alter the AV’s mechanical environment, triggering a cascade of remodeling, inflammation and calcification activities in AV tissue. Changes in mechanical environments are first sent by the endothelium that induces changes in the extracellular matrix, and triggers cell differentiation and activation. However, the molecular mechanism of this process is not very well understood. Understanding these mechanisms is critical for the development of effective medical based therapies. Recently, there have been some interesting studies on characterizing the hemodynamics associated with AV, especially in pathologies like BAV, using different experimental and numerical methods. Here, we review the current knowledge of the local AV mechanical environment and its effect on valve biology, focusing on in vitro and ex vivo approaches.

Keywords: aortic valve mechanobiology, bicuspid calcification, pressure stretch, shear stress

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Authors: Lyanne Rodriguez, Eduardo Fuente, Andrés Trostchansky, Ivan Palomo

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Cardiovascular diseases (CVD) are the leading cause of death in the world. The development of platelet-rich thrombi has been considered a trigger for acute cardiovascular events. A healthy diet, rich in fruit and vegetables, has been related to increased protection against cardiovascular events. Previous studies have observed that tomato pomace has a potent antiplatelet activity, due could be attributed to its high content of fatty acids (> 30%). It has been shown that unsaturated fatty acids can undergo endogenous intracellular nitration reactions during digestion after lipid consumption. Additionally, nitrated fatty acids (NO2-FA) can significantly reduce atherosclerotic lesion formation, inhibiting the expression of adhesion molecules on dysfunctional endothelium and platelet activation. In this work, we have proposed the nitration of fatty acids present in tomato pomace to improve its antiplatelet action. The gastric digestion of the tomato pomace allowed the nitration of the fatty acids, while by HPLC/MS/MS we were able to identify and quantify the nitrated fatty acids. The nitrated tomase extracts showed antiplatelet potential when platelets were stimulated with TRAP-6 and collagen. This activity was related to the presence of nitrated linoleic acid, which inhibited platelet activation by flow cytometry. The knowledge about the antiplatelet activity of nitrated fatty acids from tomato pomace will further develop new and more effective agents.

Keywords: cardiovascular, tomato extracts, nitrated fatty acids, antiplatelet activity

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Authors: Taha Kadir Yesin, Hanyu Liu, Zhangfan Ding, Amit Singh, Qi Tian, Yuheng Zhang, Biswajyoti Borah, Junyu Chen, Anjali P. Kusumbe

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The skeletal structure and bone marrow endothelium collectively form a critical functional unit essential for bone development, health, and aging. At the core of osteogenesis and bone formation lies the dynamic process of angiogenesis. In this study, we reveal a potent endogenous anabolic NCAM1-14.3.3. ζδ-derived- Peptide interaction, which stimulates bone angiogenesis and osteogenesis during homeostasis, aging, and age-related bone diseases. Employing high-resolution imaging and inducible cell-specific mouse genetics, our results elucidate the pivotal role of the NCAM1-14.3.3.ζδ-derived-Peptide interaction in driving the expansion of Clec14a+ angiogenic endothelial cells. Notably, Clec14a+ endothelial cells express key osteogenic factors. The NCAM1-14.3.3.ζδ-derived-Peptide interaction in osteoblasts drives osteoblast differentiation, ultimately contributing to the genesis of bone. Moreover, the NCAM1-14.3.3.ζδ-derived-Peptide interaction leads to a reduction in bone resorption. In age-associated vascular and bone loss diseases, stimulating the NCAM1-14.3.3.ζδ-derived-Peptide interaction not only promotes angiogenesis but also reverses bone loss. Consequently, harnessing the endogenous anabolic potential of the NCAM1-14.3.3.ζδ-derived-Peptide interaction emerges as a promising therapeutic modality for managing age-related bone diseases.

Keywords: endothelial cell, NCAM1, Clec14a, 14.3.3.ζδ

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Authors: Rauza Sukma Rita, Katsuya Dezaki, Yuko Maejima, Toshihiko Yada

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Oxytocin is a nine-amino acid peptide synthesized in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. Oxytocin promotes contraction of the uterus during birth and milk ejection during breast feeding. Although oxytocin receptors are found predominantly in the breasts and uterus of females, many tissues and organs express oxytocin receptors, including the pituitary, heart, kidney, thymus, vascular endothelium, adipocytes, osteoblasts, adrenal gland, pancreatic islets, and many cell lines. On the other hand, in pancreatic islets, oxytocin receptors are expressed in both α-cells and β-cells with stronger expression in α- cells. However, to our knowledge there are no reports yet about the effect of oxytocin on cytosolic calcium reaction on α and β-cell. This study aims to investigate the effect of oxytocin on α-cells and β-cells and its oscillation pattern. Islet of Langerhans from wild type mice were isolated by collagenase digestion. Isolated and dissociated single cells either α-cells or β-cells on coverslips were mounted in an open chamber and superfused in HKRB. Cytosolic concentration ([Ca2+]i) in single cells were measured by fura-2 microfluorimetry. After measurement of [Ca2+]i, α-cells were identified by subsequent immunocytochemical staining using an anti-glucagon antiserum. In β-cells, the [Ca2+]i increase in response to oxytocin was observed only under 8.3 mM glucose condition, whereas in α-cells, [Ca2+]i an increase induced by oxytocin was observed in both 2.8 mM and 8.3 mM glucose. The oscillation incidence was induced more frequently in β-cells compared to α-cells. In conclusion, the present study demonstrated that oxytocin directly interacts with both α-cells and β-cells and induces increase of [Ca2+]i and its specific patterns.

Keywords: α-cells, β-cells, cytosolic calcium concentration, oscillation, oxytocin

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Authors: Reva Sharan Thakur, Mrinalini Tiwari, Jyoti das

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Cerebral malaria-associated over expression of pro-inflammatory cytokines and chemokines ultimately results in the up-regulation of adhesion molecules in the brain endothelium leading to sequestration of mature parasitized RBCs in the brain. The high-parasitic load subsequently results in increased mortality or development of neurological symptoms within a week of infection. Studies in the human and experimental cerebral malaria have implicated the breakdown of the integrity of blood-brain barrier during the lethal course of infection, cerebral dysfunction, and fatal organ pathologies that result in multi-organ failure. In the present study, using Plasmodium berghei Anka as a mouse model and in vitro conditions, we have investigated the effect of MSCs to attenuate cerebral malaria pathogenesis by diminishing the effect of inflammation altered organ morphology, reduced parasitemia, and increased survival of the mice. MSCs are also validated for their role in preventing BBB dysfunction and reducing malarial toxins. It was observed that administration of MSCs significantly reduced parasitemia and increased survival in Pb A infected mice. It was further demonstrated that MSCs play a significant role in reversing neurological complexities associated with cerebral malaria. Infusion of MSCs in infected mice decreased hemozoin deposition; oedema, and haemorrhagic lesions in vascular organs. MSCs administration also preserved the integrity of the blood-brain barrier and reduced neural inflammation. Taken together, our results demonstrate the potential of MSCs as an emerging anti-malarial candidate.

Keywords: cerebral malaria, mesenchymal stem cells, erythropoesis, cell death

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Authors: B. Dejanova, S. Petrovska, L. Todorovska, J. Pluncevic, S. Mancevska, V. Antevska, E. Sivevska, I. Karagjozova

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Introduction: Aging process is mainly related to endothelial function which may be impaired by oxidative stress (OS). Exercise is known to be beneficial to aging process, which may improve health and prevent appearance of chronic diseases in elderly. The aim of the study was to investigate the OS markers related to exercise. Methods: A number of 80 subjects (healthy volunteers) were examined (38 male and 32 female), divided in 3 age groups: group I ≤ 30 years (n=24); group II – 31-50 years (n=24); group III - ≥ 51 year (n=32). Each group was divided to subgroups of sedentary subjects (SS) and subjects who exercise (SE). Group I: SS (n=11), SE (n=13); group II: SS (n=13), SE (n=10); group III: SS (n=23) SE (n=9). Lipid peroxidation (LP) as a fluorimetric method with thiobarbituric acid was used to estimate OS. Antioxidative status was determined by cell antioxidants such as enzymes - superoxide dismutase (SOD), glutathione peroxidase (GPx) and glucose 6 phosphate (G-6-PD); and by extra cell antioxidants such as glutathione reductase (GR), nitric oxide (NO) and total antioxidant capacity (TAC). Results: Increased values of LP were noticed along the aging process: group I – 3.30±0.3 µmol/L; group II – 3.91±0.2 µmol/L; group III – 3.94±0.8 µmol/L (p<0.05), while no statistical significance was found between male and female subjects. Statistical significance for OS was not found between SS and SE in group I as it was found in group II (p<0.05) and in group III (p<0.01). No statistical significance was found for all cell antioxidants and GR within the groups, while NO and TAC showed lower values in SS compared to SE in II (p<0.05) and in group III (p<0.05). Discussion and conclusion: Aging process showed increased OS which may be either due to impaired function of scavengers of free radicals or due to their enormous production. Well balanced exercise might be one of the factors that keep the integrity of blood vessel endothelium which slows down the aging process. Possible mechanism of exercise beneficial influence is shear stress by upregulation of genes coding for nitric oxide bioavailability. Thus, due to obtained results we may conclude that OS is found to be diminished in the subject groups who perform exercise.

Keywords: oxidative stress, aging process, exercise, endothelial function

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Authors: Ying Sun, Biao Cheng, Qing Lu, Xuefei Tao, Xiaoyu Lai, Cheng Guo, Dan Wang

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Fibrinogen-like protein 2 (FGL2) has recently been identified to play an important role in inflammatory diseases such as atherosclerosis through a thrombin-dependent manner. Here, a murine monoclonal antibody was raised against the critical residue Ser(89) of FGL2, and the effects of the anti-FGL2 mAb (SPF89) were analyzed in human umbilical vein endothelial cells (HUVECs) and THP-1 cells. Firstly, it was proved that SPF89, which belongs to the IgG1 subtype with a KD value of 44.5 pM, could specifically show the expression levels of protein FGL2 in different cell lines of known target gene status. The lipopolysaccharide (LPS)-mediated endothelial cell proliferation was significantly inhibited with a decline of phosphorylation nuclear factor-κB (NF-κB) in a dose-dependent manner after SPF89 treatment. Furthermore, SPF89 reduced LPS-induced expression of adhesion molecules and inflammatory cytokines such as vascular cell adhesion molecule-1, tumor necrosis factor-α, Matrix metalloproteinase MMP-2, Integrin αvβ3, and interleukin-6 in HUVECs. In macrophage-like THP-1 cells, SPF89 effectively inhibited LPS and low-density lipoprotein-induced foam cell formation. However, these anti-inflammatory and anti-atherosclerotic effects of anti-FGL2 mAb in HUVECs and THP-1 cells were significantly reduced after treatment with an NF-κB inhibitor PDTC. All the above suggest, by efficiently inhibiting LPS-induced pro-inflammatory effects in vascular endothelial cells by attenuating NF-κB dependent pathway, the new anti-FGL2 mAb SPF89 could to be a potential therapeutic candidate for protecting the vascular endothelium against inflammatory diseases such as atherosclerosis. This work was supported by the Program of Sichuan Science and Technology Department (2017FZ0069) and Collaborative Innovation Program of Sichuan for Elderly Care and Health(YLZBZ1511).

Keywords: monoclonal antibody, fibrinogen like protein 2, inflammation, endothelial cells

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Authors: Yi Sun, George Ed Rainger, Steve P. Watson

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Background: Beyond the classical roles in haemostasis and thrombosis, platelets are important in the initiation and development of various thrombo-inflammatory diseases. In atherosclerosis and deep vein thrombosis, for example, platelets bridge monocytes with endothelium and form heterotypic aggregates with monocytes in the circulation. This can alter monocyte phenotype by inducing their activation, stimulating adhesion and migration. These interactions involve cell surface receptor-ligand pairs on both cells. This list is likely incomplete as new interactions of importance to platelet biology are continuing to be discovered as illustrated by our discovery of PEAR-1 binding to FcεR1α. Results: We have developed a highly sensitive avidity-based assay to identify novel extracellular interactions among 126 recombinantly-expressed platelet cell surface and secreted proteins involved in platelet aggregation. In this study, we will use this method to identify novel platelet-monocyte interactions. We aim to identify ligands for orphan receptors and novel partners of well-known proteins. Identified interactions will be studied in preliminary functional assays to demonstrate relevance to the inflammatory processes supporting atherogenesis. Conclusions: Platelet-monocyte interactions are essential for the development of thromboinflammatory disease. Up until relatively recently, technologies only allow us to limit our studies on each individual protein interaction at a single time. These studies propose for the first time to study the cell surface platelet-monocyte interactions in a systematic large-scale approach using a reliable screening method we have developed. If successful, this will likely to identify previously unknown ligands for important receptors that will be investigated in details and also provide a list of novel interactions for the field. This should stimulate studies on developing alternative therapeutic strategies to treat vascular inflammatory disorders such as atherosclerosis, DVT and sepsis and other clinically important inflammatory conditions.

Keywords: membrane proteins, large-scale screening, platelets, recombinant expression

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Authors: Lotfollah Rezagholizadeh

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Background and Aim: Diabetes mellitus is related to high mortality and morbidity caused by the early development of atherosclerosis correlated to diabetic macroangiopathy. The endothelium-derived vasodilator, nitric oxide (NO) has been implicated in the development of vascular complications via the regulation of blood flow, and various antiatherosclerotic actions. Patients with type 2 diabetes (T2D) have a decreased level of endothelial nitric oxide release. In this study we aimed to examine the effect of aquatic seed extract of Cichorium intybus L. (chicory) and metformin (a known prescription drug for diabetes) on NO levels in T2D rats. Methods: Five groups of adult male Wistar rats were used (n=6): Non-diabetic controls without extract treatment (Control), Non-diabetic controls with extract treatment (Chicory-control), T2D rats without extract treatment (NIA/STZ), T2D rats treated with the extract (Chicory-NIA/STZ), and T2D groups that received metformin (100 mg/kg) but no extract (Metformin-NIA/STZ). T2D was induced with intraperitoneal (i.p) injection of niacinamide (NIA, 200 mg/kg), 15 min after an i.p administration of streptozotocin (STZ, 55 mg/kg). Lyophilized chicory extract (125 mg/kg) was dissolved in 0.2 ml normal saline and administered one dose a day. The experiments lasted for 3 weeks after the diabetes induction. NO analysis was performed by assay based on the Griess reaction. Data were reported as the mean ± SD and statistical analysis was performed by ANOVA. Results: Serum nitric oxide levels decreased significantly in NIA/STZ group compared with Control and Chicory-control. Treatment with chicory extract caused a significant increase in serum levels of NO in Chicory-NIA/STZ group compare to NIA/STZ group (p<05). Metformin-NIA/STZ group did not show considerable difference when compared with NIA/STZ, with respect to NO levels. In a group of rats made diabetic by STZ alone (type 1 diabetic rats, T1D), chicory did not have a significant ameliorating effect. Conclusion: In this study, we clearly showed a relationship between low serum nitric oxide levels and diabetes mellitus in rats. The increase in serum nitric oxide by chicory extract is an indication of antiatherogenic effect of this plant. Chicory seed extract was more efficient than metformin in improving the NO levels in NO-deficient T2D diabetic rats.

Keywords: type 2 diabetes mellitus, nitric oxide, chicory, metformin

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Authors: Kamsiah Jaarin, Yusof Kamisah, Faizah Othman Nurul Akmal Muhammad, Zakiah Jubri, Qodriyah Mohd Saad, Srijit Das

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Forty (40) normotensive adult male Sprague-Dawley rats aged three months weighing 180-200 g were divided into 4 groups with 10 rats per group: (1) normotensive control; (2) hypertensive rats; (3) hypertensive rats treated with Nigella sativa (2.5 ml/kg/day); and (4) hypertensive rats treated with nicardipine (5 mg/kg/day). After acclimatization, the hypertensive rats of the group 2, 3 and 4 were induced to be hypertensive by giving NW–nitro-L-arginine methyl ester (L-NAME; 30 mg/kg/day) in their drinking water for consecutive 7 days. After one week, rats in the group 3 were given a daily oral dose of 2.5 ml/kg/day of Nigella sativa (NS) by oral gavage. Rats in the group 4 were given nicardipine (5 mg/kg/day) via oral gavages. All rats in this study received L-NAME continuously throughout the treatment duration. The blood pressure will be measured pre-treatment and weekly for 8 weeks using power lab. Blood was taken before and at the end of study for measurement of nitric oxide. At the end of 8 weeks, the rats are sacrificed and descending thoracic aorta was disserted for measurement of vascular reactivity, and intracellular adhesion molecules (ICAM-1) and vascular cell adhesion molecules (VCAM-1). Nigella sativa reduced both systolic and diastolic BP compared to control and L-name group. The BP lowering effect of NS was comparable to nicardipine a calcium antagonist. The blood pressure lowering effect of NS was accompanied with an increasing relaxation response to nitroprusside and acetylcholine and reducing vasoconstriction response to epinephrine. L-NAME and nicardipine on the other hand, reduced plasma nitric oxide concentration. In contrast, NS increased NO concentration. However, Nigella sativa had no significant effect on aortic VCAM- 1 and ICAM-1 expression. In conclusion; Nigella sativa oil reduces both systolic and diastolic blood pressure in L-NAME treated rats. The antihypertensive effect of NS was comparable to nicardipine. The BP lowering effect may be mediated via stimulating nitric oxide release from vascular endothelium.

Keywords: Nigella sativa, ICAM, VCAM, blood pressure, vascular reactivity

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Authors: Amany Ragab, Nashwa Khairat Abousamra, Omayma Saleh, Asmaa Higazy

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Insulin resistance syndrome has been shown to be associated with many coagulation and fibrinolytic proteins and these associations suggest that some coagulation and fibrinolytic proteins have a role in atherothrombotic disorders. This study was conducted to determine the levels of some of the haemostatic parameters in subjects having metabolic syndrome and to correlate these values with the anthropometric and metabolic variables associated with this syndrome. The study included 46 obese non diabetic subjects of whom 28 subjects(group1) fulfilled the ATP III criteria of the metabolic syndrome and 18 subjects (group2) did not have metabolic syndrome as well as 14 lean subjects (group 3) of matched age and sex as a control group. Clinical and laboratory evaluation of the study groups stressed on anthropometric measurements (weight, height, body mass index, waist circumference, and sagittal abdominal diameter), blood pressure, and laboratory measurements of fasting plasma glucose, fasting insulin, serum lipids, tissue plasminogen activator (t-PA), antithrombin III activity (ATIII), protein C and von Willebrand factor (vWf) antigen. There was significant increase in the concentrations of t-PA and vWf antigens in subjects having metabolic syndrome (group 1) in comparison to the other groups while there were non-significant changes in the levels of protein C antigen and AT III activity. Both t-PA and vWf showed significant correlation with HOMA-IR as a measure of insulin sensitivity. The t-PA showed also significant correlation with most of the variables of metabolic syndrome including waist circumference, BMI, systolic blood pressure, fasting plasma glucose, fasting insulin, and HDL cholesterol. On the other hand, vWf showed significant correlations with fasting plasma glucose, fasting insulin and sagital abdominal diameter, with non-significant correlations with the other variables. Haemostatic and fibrinolytic parameters should be included in the features and characterization of the insulin resistance syndrome. t-PA and vWf antigens concentrations were increased in subjects with metabolic syndrome and correlated with the HOMA-IR measure of insulin sensitivity. Taking into consideration that both t-PA and vWf are mainly released from vascular endothelium, these findings could be an indicator of endothelial dysfunction in that group of subjects.

Keywords: insulin resistance, obesity, metabolic syndrome, coagulation

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Authors: Mohamed Salah El-Din Mahmoud, Heba Radi Atta Allah

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Background: Our study assesses the safety profile and efficacy of toric Implantable Collamer Lens (T-ICL) and toric implantable phakic contact lens (IPCL) for the correction of high myopia with astigmatism. Methods: A prospective interventional randomized comparative study included 60 myopic eyes divided into 2 groups, group A including 30 eyes that were implanted with T-ICL, and group B including 30 eyes that were implanted with toric IPCL. The refractive results, visual acuity, corneal endothelial cell count, and intraocular pressure (IOP) were evaluated at baseline and at 1, 6, and 9 months post-surgery. Any complications either during or after surgery were assessed. Results: A significant reduction in both spherical and cylindrical refractive errors with good predictability was reported in both groups compared with preoperative values. Regarding the predictability, In T-ICL group (A), the median spherical and cylindrical errors were significantly improved from (-10 D & -4.5 D) pre-operatively to (-0.25 D & - 0.3 D) at the end of 9 months follow up period. Similarly, in the toric IPCL group (B), the median spherical and cylindrical errors were significantly improved from (-11 D & -4.5 D) pre-operatively to (-0.25 D & - 0.3 D) at the end of 9 months follow up period. A statistically significant improvement of UCDVA at 9 months postoperatively was found in both groups, as median preoperative Log Mar UCDVA was 1.1 and 1.3 in groups A and B respectively, which was significantly improved to 0.2 in both groups at the end of follow-up period. Regarding IOP, no significant difference was found between both groups, either pre-operatively or during the postoperative period. Regarding the endothelial count, no significant differences were found during the pre-operative and postoperative follow-up periods between the two groups. Fortunately, no intra or postoperative complications as cataract, keratitis or lens decentration had occurred. Conclusions: Toric IPCL is a suitable alternative to T-ICL for the management of high myopia with astigmatism, especially in developing countries, as it is cheaper and easier for implantation than T-ICL. However, data over longer follow-up periods are needed to confirm its safety and stability.

Keywords: T-ICL, Toric IPCL, IOP, corneal endothelium

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Authors: Semyachkina-Glushkovskaya Oxana, Fedosov Ivan, Blokhina Inna, Terskov Andrey, Evsukova Arina, Elovenko Daria, Adushkina Viktoria, Dubrovsky Alexander, Jürgen Kurths

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In modern neurobiology, sleep is considered a novel biomarker and a promising therapeutic target for brain diseases. This is due to recent discoveries of the nighttime activation of the brain lymphatic system (BLS), playing an important role in the removal of wastes and toxins from the brain and contributes neuroprotection of the central nervous system (CNS). In our review, we discuss that night stimulation of BLS might be a breakthrough strategy in a new treatment of Alzheimer’s and Parkinson’s disease, stroke, brain trauma, and oncology. Although this research is in its infancy, however, there are pioneering and promising results suggesting that night transcranial photostimulation (tPBM) stimulates more effectively lymphatic removal of amyloid-beta from mouse brain than daily tPBM that is associated with a greater improvement of the neurological status and recognition memory of animals. In our previous study, we discovered that tPBM modulates the tone and permeability of the lymphatic endothelium by stimulating NO formation, promoting lymphatic clearance of wastes and toxins from the brain tissues. We also demonstrate that tPBM can also lead to angio- and lymphangiogenesis, which is another mechanism underlying tPBM-mediated stimulation of BLS. Thus, photo-augmentation of BLS might be a promising therapeutic target for preventing or delaying brain diseases associated with BLS dysfunction. Here we present pioneering technology for simultaneous tPBM in humans and sleep monitoring for stimulation of BLS to remove toxins from CNS and modulation of brain immunity. The wireless-controlled gadget includes a flexible organic light-emitting diode (LED) source that is controlled directly by a sleep-tracking device via a mobile application. The designed autonomous LED source is capable of providing the required therapeutic dose of light radiation at a certain region of the patient’s head without disturbing of sleeping patient. To minimize patients' discomfort, advanced materials like flexible organic LEDs were used. Acknowledgment: This study was supported by RSF project No. 23-75-30001.

Keywords: brain diseases, brain lymphatic system, phototherapy, sleep

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Authors: Xiaobo Wang, Zhen-Yu Chen

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Higher plasma total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) are independent risk factors of cardiovascular disease while high-density lipoprotein cholesterol (HDL-C) is protective. Vitamin D is well-known for its regulatory role in calcium homeostasis. Its potential important role in cardiovascular disease has recently attracted much attention. This study was conducted to investigate effects of different dosage of calcitriol on plasma lipoprotein profile and the underlying mechanism. Sixty male Syrian Golden hamsters were randomly divided into 6 groups: no-cholesterol control (NCD), high-cholesterol control (HCD), groups with calcitriol supplementation at 10/20/40/80ng/kg body weight (CA, CB, CC, CD), respectively. Calcitriol in medium-chain triacylglycerol (MCT) oil was delivered to four experimental groups via oral gavage every other day, while NCD and HCD received MCT oil in the equivalent amount. NCD hamsters were fed with non-cholesterol diet while other five groups were maintained on diet containing 0.2% cholesterol to induce a hypercholesterolemic condition. The treatment lasts for 6 weeks followed by sample collection after hamsters sacrificed. Four experimental groups experienced a reduction in average food intake around 11% compared to HCD with slight decrease in body weight (not exceeding 10%). This reduction reflects on the deceased relative weights of testis, epididymal and perirenal adipose tissue in a dose-dependent manner. Plasma calcitriol levels were measured and was corresponding to oral gavage. At the end of week 6, lipoprotein profiles were improved with calcitriol supplementation with TC, non-HDL-C and plasma triglyceride (TG) decreased in a dose-dependent manner (TC: r=0.373, p=0.009, non-HDL-C: r=0.479, p=0.001, TG: r=0.405, p=0.004). Since HDL-C of four experiment groups showed no significant difference compared to HCD, the ratio of nHDL-C to HDL-C and HDL-C to TC had been restored in a dose-dependent manner. For hamsters receiving the highest level of calcitriol (80ng/kg) showed a reduction of TC by 11.5%, nHDL-C by 24.1% and TG by 31.25%. Little difference was found among six groups on the acetylcholine-induced endothelium-dependent relaxation or contraction of thoracic aorta. To summarize, calcitriol supplementation in hamster at maximum 80ng/kg body weight for 6 weeks lead to an overall improvement in plasma lipoprotein profile with decreased TC and TG level. The molecular mechanism of its effects is under investigation.

Keywords: cholesterol, vitamin D, calcitriol, hamster

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Authors: Laura M. Hovsepyan, Gayane S. Ghazaryan, Hasmik V. Zanginyan

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Hypertension (HD) is the most common cardiovascular pathology that causes disability and mortality in the working population. Most often, heart failure (HF), which is based on myocardial remodeling, leads to death in hypertension. Recently, endothelial dysfunction (EDF) or a violation of the functional state of the vascular endothelium has been assigned a significant role in the structural changes in the myocardium and the occurrence of heart failure in patients with hypertension. It has now been established that tissues affected by inflammation form increased amounts of superoxide radical and NO, which play a significant role in the development and pathogenesis of various pathologies. They mediate inflammation, modify proteins and damage nucleic acids. The aim of this work was to study the processes of oxidative modification of proteins (OMP) and the production of nitric oxide in hypertension. In the experimental work, the blood of 30 donors and 33 patients with hypertension was used. For the quantitative determination of OMP products, the based on the reaction of the interaction of oxidized amino acid residues of proteins and 2,4-dinitrophenylhydrazine (DNPH) with the formation of 2,4-dinitrophenylhydrazones, the amount of which was determined spectrophotometrically. The optical density of the formed carbonyl derivatives of dinitrophenylhydrazones was recorded at different wavelengths: 356 nm - aliphatic ketone dinitrophenylhydrazones (KDNPH) of neutral character; 370 nm - aliphatic aldehyde dinirophenylhydrazones (ADNPH) of neutral character; 430 nm - aliphatic KDNFG of the main character; 530 nm - basic aliphatic ADNPH. Nitric oxide was determined by photometry using Grace's solution. Adsorption was measured on a Thermo Scientific Evolution 201 SF at a wavelength of 546 nm. Thus, the results of the studies showed that in patients with arterial hypertension, an increased level of nitric oxide in the blood serum is observed, and there is also a tendency to an increase in the intensity of oxidative modification of proteins at a wavelength of 270 nm and 363 nm, which indicates a statistically significant increase in aliphatic aldehyde and ketone dinitrophenylhydrazones. The increase in the intensity of oxidative modification of blood plasma proteins in the studied patients, revealed by us, actually reflects the general direction of free radical processes and, in particular, the oxidation of proteins throughout the body. A decrease in the activity of the antioxidant system also leads to a violation of protein metabolism. The most important consequence of the oxidative modification of proteins is the inactivation of enzymes.

Keywords: hypertension (HD), oxidative modification of proteins (OMP), nitric oxide (NO), oxidative stress

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Authors: Gabriela C. Caldas, Fernanda C. Jacome, Arthur da C. Rasinhas, Ortrud M. Barth, Flavia B. dos Santos, Priscila C. G. Nunes, Yuli R. M. de Souza, Pedro Paulo de A. Manso, Marcelo P. Machado, Debora F. Barreto-Vieira

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The establishment of animal models for studies of DENV infections has been challenging, since circulating epidemic viruses do not naturally infect nonhuman species. Such studies are of great relevance to the various areas of dengue research, including immunopathogenesis, drug development and vaccines. In this scenario, the main objective of this study is to verify possible morphological changes, as well as the presence of antigens and viral RNA in lung samples from BALB/c mice experimentally infected with an epidemic and non-neuroadapted DENV-3 strain. Male BALB/c mice, 2 months old, were inoculated with DENV-3 by intravenous route. After 72 hours of infection, the animals were euthanized and the lungs were collected. Part of the samples was processed by standard technique for analysis by light and transmission electronic microscopies and another part was processed for real-time PCR analysis. Morphological analyzes of lungs from uninfected mice showed preserved tissue areas. In mice infected with DENV-3, the analyzes revealed interalveolar septum thickening with presence of inflammatory infiltrate, foci of alveolar atelectasis and hyperventilation, bleeding foci in the interalveolar septum and bronchioles, peripheral capillary congestion, accumulation of fluid in the blood capillary, signs of interstitial cell necrosis presence of platelets and mononuclear inflammatory cells circulating in the capillaries and/or adhered to the endothelium. In addition, activation of endothelial cells, platelets, mononuclear inflammatory cell and neutrophil-type polymorphonuclear inflammatory cell evidenced by the emission of cytoplasmic membrane prolongation was observed. DEN-like particles were seen in the cytoplasm of endothelial cells. The viral genome was recovered from 3 in 12 lung samples. These results demonstrate that the BALB / c mouse represents a suitable model for the study of the histopathological changes induced by DENV infection in the lung, with tissue alterations similar to those observed in human cases of DEN.

Keywords: BALB/c mice, dengue, histopathology, lung, ultrastructure

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Authors: Lijuan Li

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Nitric oxide (NO) has become a fascinating entity in biological chemistry over the past few years. It is a gaseous lipophilic radical molecule that plays important roles in several physiological and pathophysiological processes in mammals, including activating the immune response, serving as a neurotransmitter, regulating the cardiovascular system, and acting as an endothelium-derived relaxing factor. NO functions in eukaryotes both as a signal molecule at nanomolar concentrations and as a cytotoxic agent at micromolar concentrations. The latter arises from the ability of NO to react readily with a variety of cellular targets leading to thiol S-nitrosation, amino acid N-nitrosation, and nitrosative DNA damage. Nitric oxide can readily bind to metals to give metal-nitrosyl (M-NO) complexes. Some of these species are known to play roles in biological NO storage and transport. These complexes have different biological, photochemical, or spectroscopic properties due to distinctive structural features. These recent discoveries have spawned a great interest in the development of transition metal complexes containing NO, particularly its iron complexes that are central to the role of nitric oxide in the body. Spectroscopic evidence would appear to implicate species of “Fe(NO)2+” type in a variety of processes ranging from polymerization, carcinogenesis, to nitric oxide stores. Our research focuses on isolation and structural studies of non-heme iron nitrosyls that mimic biologically active compounds and can potentially be used for anticancer drug therapy. We have shown that reactions between Fe(NO)2(CO)2 and a series of imidazoles generated new non-heme iron nitrosyls of the form Fe(NO)2(L)2 [L = imidazole, 1-methylimidazole, 4-methylimidazole, benzimidazole, 5,6-dimethylbenzimidazole, and L-histidine] and a tetrameric cluster of [Fe(NO)2(L)]4 (L=Im, 4-MeIm, BzIm, and Me2BzIm), resulted from the interactions of Fe(NO)2 with a series of substituted imidazoles was prepared. Recently, a series of sulfur bridged iron di nitrosyl complexes with the general formula of [Fe(µ-RS)(NO)2]2 (R = n-Pr, t-Bu, 6-methyl-2-pyridyl, and 4,6-dimethyl-2-pyrimidyl), were synthesized by the reaction of Fe(NO)2(CO)2 with thiols or thiolates. Their structures and properties were studied by IR, UV-vis, 1H-NMR, EPR, electrochemistry, X-ray diffraction analysis and DFT calculations. IR spectra of these complexes display one weak and two strong NO stretching frequencies (νNO) in solution, but only two strong νNO in solid. DFT calculations suggest that two spatial isomers of these complexes bear 3 Kcal energy difference in solution. The paramagnetic complexes [Fe2(µ-RS)2(NO)4]-, have also been investigated by EPR spectroscopy. Interestingly, the EPR spectra of complexes exhibit an isotropic signal of g = 1.998 - 2.004 without hyperfine splitting. The observations are consistent with the results of calculations, which reveal that the unpaired electron dominantly delocalize over the two sulfur and two iron atoms. The difference of the g values between the reduced form of iron-sulfur clusters and the typical monomeric di nitrosyl iron complexes is explained, for the first time, by of the difference in unpaired electron distributions between the two types of complexes, which provides the theoretical basis for the use of g value as a spectroscopic tool to differentiate these biologically active complexes.

Keywords: di nitrosyl iron complex, metal nitrosyl, non-heme iron, nitric oxide

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Authors: Ruth Goldschmidt, Vyacheslav Kalchenko, Lilah Agemy, Rachel Elmoalem, Avigdor Scherz

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Vascular Targeted Photodynamic therapy (VTP) is a new modality for selective cancer treatment that leads to the complete tumor ablation. A photosensitizer, a bacteriochlorophyll derivative in our case, is first administered to the patient and followed by the illumination of the tumor area, by a near-IR laser for its photoactivation. The photoactivated drug releases reactive oxygen species (ROS) in the circulation, which reacts with blood cells and the endothelium leading to the occlusion of the blood vasculature. If the blood vessels are only partially closed, the tumor may recover, and cancer cells could survive. On the other hand, excessive treatment may lead to toxicity of healthy tissues nearby. Simultaneous VTP monitoring and image processing independent of the photoexcitation laser has not yet been reported, to our knowledge. Here we present a method for blood flow monitoring, using a real-time laser speckle imaging (RTLSI) in the tumor during VTP. We have synthesized over the years a library of bacteriochlorophyll derivatives, among them WST11 and STL-6014. Both are water soluble derivatives that are retained in the blood vasculature through their partial binding to HSA. WST11 has been approved in Mexico for VTP treatment of prostate cancer at a certain drug dose, and time/intensity of illumination. Application to other bacteriochlorophyll derivatives or other cancers may require different treatment parameters (such as light/drug administration). VTP parameters for STL-6014 are still under study. This new derivative mainly differs from WST11 by its lack of the central Palladium, and its conjugation to an Arg-Gly-Asp (RGD) sequence. RGD is a tumor-specific ligand that is used for targeting the necrotic tumor domains through its affinity to αVβ3 integrin receptors. This enables the study of cell-targeted VTP. We developed a special RTLSI module, based on Labview software environment for data processing. The new module enables to acquire raw laser speckle images and calculate the values of the laser temporal statistics of time-integrated speckles in real time, without additional off-line processing. Using RTLSI, we could monitor the tumor’s blood flow following VTP in a CT26 colon carcinoma ear model. VTP with WST11 induced an immediate slow down of the blood flow within the tumor and a complete final flow arrest, after some sporadic reperfusions. If the irradiation continued further, the blood flow stopped also in the blood vessels of the surrounding healthy tissue. This emphasizes the significance of light dose control. Using our RTLSI system, we could prevent any additional healthy tissue damage by controlling the illumination time and restrict blood flow arrest within the tumor only. In addition, we found that VTP with STL-6014 was the most effective when the photoactivation was conducted 4h post-injection, in terms of tumor ablation success in-vivo and blood vessel flow arrest. In conclusion, RTSLI application should allow to optimize VTP efficacy vs. toxicity in both the preclinical and clinical arenas.

Keywords: blood vessel occlusion, cancer treatment, photodynamic therapy, real time imaging

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