Calcineurin/NFAT Activation-Dependence of Leptin Synthesis and Vascular Growth in Response to Mechanical Stretch

Background and Aims- Hypertension and obesity are important risk factors of cardiovascular disease. They are both associated with high leptin levels and have been shown to promote vascular hypertrophy, through the RhoA/ROCK and ERK1/2 phosphorylation. Calcineurin/NFAT activation also induces vascular hypertrophy by upregulating various genes. This study aimed to decipher whether a crosstalk exists between the RhoA/ROCK pathway, Ca+2/calcineurin/NFAT pathway, and ERK1/2 phosphorylation in the process of mechanical stretch-induced vascular smooth muscle cell (VSMC) hypertrophy and leptin synthesis. Methods and Results- Rat portal vein (RPV) organ culture was used to investigate the effect of mechanical stretch and exogenous leptin (3.1 nM) on VSMC hypertrophy and leptin synthesis. Results showed that stretching the RPV significantly upregulated leptin secretion, mRNA and protein expression, which were inhibited by the calcium channel blocker nifedipine (10 μM), the selective calcineurin inhibitor FK506 (1 nM) and the ERK1/2 inhibitor PD98059 (1 μM). The transcription inhibitor actinomycin D (0.1M) and the translation inhibitor cycloheximide (1 mM) significantly decreased stretch-induced leptin protein expression. Mechanical stretch or leptin caused an increase in wet weight changes and protein synthesis, considered as hypertrophic markers, while they were inhibited by FK506 (0.1 nM; 1 nM). In addition, stretch or exogenous leptin significantly increased calcineurin activity and MCIP1 expression whereas leptin induced NFAT nuclear translocation in VSMCs. Moreover, in response to stretch or exogenous leptin, the Rho inhibitor C3 exoenzyme (30 ng/mL), the ROCK inhibitor Y-27632 (10 μM), and the actin depolymerization agents Latrunculin B (50 nM) and cytochalasin D (1 μM) reduced calcineurin activation and NFAT nuclear translocation. ERK1/2 phosphorylation was inhibited by FK506 and C3. Conclusions- Mechanical stretch-induced VSMC hypertrophy and leptin synthesis and secretion is mediated by Ca2+/calcineurin/NFAT activation. RhoA/ROCK and ERK1/2 activation are critical for mechanical stretch-induced calcineurin activation

Evaluation of Antioxidant, Anticancer and Immunomodulatory Activities of Wild Carrot (Daucus carota ssp. carota) Oil and its Fractions.

Daucus carota L. ssp. carota, also known as wild carrot, has been used in traditional medicine in Lebanon to treat several diseases including cancer. The aim of the present study was to characterize the chemical constituents of D. carota oil extract (DCOE) and evaluate its anticancer activity. DCOE was chromatographed on silica gel column and four fractions were obtained. GC-MS and HPLC analysis of DCOE and fractions showed the presence of several sesquiterpenes and phenolic compounds. A major finding was the identification of 2-himachalene-6-ol, a sesquiterpene reported for the first time from wild carrot plant species. In vitro antioxidant activity of DCOE and its fractions was demonstrated and correlated to the phenolic and flavonoid contents. In vivo antioxidant and hepatoprotective activities of DCOE fractions was also observed in CCl14-induced hepatotoxicity in mice. The anticancer effects of DCOE and its fractions were evaluated against several human cancer cell lines and found to exhibit dose dependent anti-proliferative activities, with F1 and F2 demonstrating the highest cytotoxicity. The inhibition of cell growth by F1 or F2 fractions was mainly due to cell cycle arrest and apoptotic cell death as assessed by flow cytometry analysis and western blot analysis of several pro-apoptotic and anti-apoptotic proteins. Additionally, the induction of apoptosis was mediated through the inhibition of both MAPK/Erk and P13K/Akt pathways in human colon cancer cells (HT-29) and only through MAPK/Erk pathway in human breast cancer cells (MDA-MB-231). The antitumour promoting effect of F2 was illustrated by significant decrease in tumour incidence, yield and volume in DMBA/TPA- induced mouse skin carcinogenesis. Also, DCOE and fractions exhibited in vivo immunomodulatory effects through enhancing the release of immunomediators. In conclusion, the present study confirms the anticancer activity of wild carrot which can be considered as a potential remedy against various types of cancer with limited toxicity

Evaluation of Antioxidant, Anticancer and Immunomodulatory Activities of Wild Carrot (Daucus carota ssp. carota) Oil and its Fractions.

Daucus carota L. ssp. carota, also known as wild carrot, has been used in traditional medicine in Lebanon to treat several diseases including cancer. The aim of the present study was to characterize the chemical constituents of D. carota oil extract (DCOE) and evaluate its anticancer activity. DCOE was chromatographed on silica gel column and four fractions were obtained. GC-MS and HPLC analysis of DCOE and fractions showed the presence of several sesquiterpenes and phenolic compounds. A major finding was the identification of 2-himachalene-6-ol, a sesquiterpene reported for the first time from wild carrot plant species. In vitro antioxidant activity of DCOE and its fractions was demonstrated and correlated to the phenolic and flavonoid contents. In vivo antioxidant and hepatoprotective activities of DCOE fractions was also observed in CCl14-induced hepatotoxicity in mice. The anticancer effects of DCOE and its fractions were evaluated against several human cancer cell lines and found to exhibit dose dependent anti-proliferative activities, with F1 and F2 demonstrating the highest cytotoxicity. The inhibition of cell growth by F1 or F2 fractions was mainly due to cell cycle arrest and apoptotic cell death as assessed by flow cytometry analysis and western blot analysis of several pro-apoptotic and anti-apoptotic proteins. Additionally, the induction of apoptosis was mediated through the inhibition of both MAPK/Erk and P13K/Akt pathways in human colon cancer cells (HT-29) and only through MAPK/Erk pathway in human breast cancer cells (MDA-MB-231). The antitumour promoting effect of F2 was illustrated by significant decrease in tumour incidence, yield and volume in DMBA/TPA- induced mouse skin carcinogenesis. Also, DCOE and fractions exhibited in vivo immunomodulatory effects through enhancing the release of immunomediators. In conclusion, the present study confirms the anticancer activity of wild carrot which can be considered as a potential remedy against various types of cancer with limited toxicity

UVB damage onset and progression 24Â h post exposure in human-derived skin cells

The focus of this research was on UVB radiation (280â320 nm) responsible for cellular changes in skin of acute and chronically exposed individuals. This study investigated the acute cellular damages triggered by UVB exposure of cultured human fibroblasts and keratinocyte cells immediately and 24 h post exposure in order to understand damage onset and progression. The study evaluated a number of cellular parameters including mitochondria, lysosomes, cell membrane, DNA damages as well as pro and anti-apoptotic protein expression levels. Cellular organelle damages were assessed by a battery of in vitro toxicological assays using MTS and Neutral red cytotoxicity assays. Cell membrane damages were also assessed by measuring lactate dehydrogenase (LDH) enzyme leakage from UVB exposed cells. Lastly DNA damages was assessed using the comet assay while protein expression was evaluated using Western Blot.In this study we reported in all our assay systems (MTS, NR and LDH) that cellular damages were UVB dose dependent with damages amplified 24 h post exposure. Our results also indicated that incubation of exposed cells for a period of 24 h increased the sensitivity of the assay systems used. The increased sensitivity in detecting early cytotoxic damages was manifested though organelle damage measurement at very low doses which were not manifested immediately post exposure. The data also indicated that HaCaT cells were most sensitive in detecting UVB triggered damages immediately and 24 h post exposure using the MTS assay. We also established upregulation and downregulation of various apoptotic proteins at various time points post exposure. The presented data clearly indicated the need for a comprehensive assessment of UVB damages 4 and 24 h post exposure due to the different assay sensitivities in addition to various signaling mechanisms activated at different time points post exposure. Keywords: HaCaT, UVB, MTS, Neutral red, LDH, Apoptosis, Comet assay, MAPK pathwa

Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method

The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels) and reduced into smaller fat pieces of around 1–3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in “Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs) into osteoblastic lineage are passage dependent” [1]. Keywords: Adipose tissue, mesenchymal stromal cell, cell culture, doubling tim